This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tandem mass spectrometry has the potential to solve the problem of rapid sequencing of the heparin and heparan sulfate (HS) carbohydrate classes. This is of critical importance to biomedicine because of the many growth factor-receptor interactions that depend on expression of HS in a tissue and developmentally regulated manner. To date collisional activated dissociation of HS has been limited by undesirable sulfate losses. Activated electron dissociation techniques show potential for HS but are limited by low efficiency and concomitant low sensitivity. The goal of this project is to develop reliable, sensitive and high throughput methods for HS sequencing. HS from bovine aorta, lung and porcine intestinal mucosa were exhaustively digested with heparin Lyase III. Oligosaccharides were then fractionated using a size exclusion column. Only the degree of polymerization (dp) 4, 6 and 8 fractions were analyzed by tandem mass spectrometry. A ThermoFisher Scientific LTQ Orbitrap Discovery mass spectrometer coupled with a Triversa Nanomate system was operated in negative ion mode. The different samples were sprayed in 50/50 methanol/water. Tandem mass spectrometry data were automatically acquired in the LTQ with a resolution of 15000, the window of isolation was 5u wide and the collision energy was maintained at 15%.